PROJECT SUMMARY Hydrogen peroxide is hazardous, though common side-productive of aerobic respiration; and many gram negative bacteria synthesize cytochrome c peroxidase (bCCP) enzymes to detoxify the peroxide by reducing it to water. bCCPs are a large family of enzymes that are marked by (at least two) c-type heme cofactors that are covalently attached into the protein backbone by the action of the cytochrome c maturation machinery. bCCPs encompass ?true? bCCP enzymes that engage primarily in the chemistry implied by their name, but also other enzymes such as MauG, which is responsible for the oxidative coupling of two tyrosine side-chains in the methylamine dehydrogenase precursor (pre-MADH) to make functional MADH, that bears a unique tryptophanyl tryptophane quinone (TTQ) cofactor. The structural bases for the differences of fundamental chemistry displayed by bCCP and MauG are poorly understood to date, but are certainly linked to the differences in the redox chemistry of conserved heme cofactors. In true bCCP enzymes, peroxidatic heme (FeL) is reponsible for peroxide binding and reduction; while electrons are transmitted to the active site via a heme that is over 500 mV higher in potential (FeH). In MauG, the potentials of the two heme cofactors are quite close, though how this trait is linked to reactivity is enigmatic. This proposal will elucidate the structure-function relationships of the bCCP family, including the distinctive chemistry of MauG vs. bCCP, and the molecular determinants for activation found within the bCCP family. Further, we have employed bioinformatics approaches to reveal new branches of the bCCP family; these include enzymes like YhjA found in enteric pathogens such as E. coli. YhjA and its paralogs possess a third, N-terminal heme binding domain, with an unknown function. A second new class of enzymes displays sequence-based traits that make it appear to be a MauG paralog, but they are found predominantly in the lung pathogen Burkholderia and not in a mau operon. Here will determine the chemistry of these novel bCCP in order to better understand the range of chemistry of the entire family. The objectives of this proposal are to reveal the stucture-function relationship amongst the members of the bCCP family, and to develop new models for the novel chemistries of enzymes such as Yhja, and the previously unreported Burkholderia bCCPs. To do so, a combination of enzymology, protein electrochemistry, and various spectroscopies (EPR, Mssbauer) and x-ray crystallography will be used to paint a portrait of the reactivities and conformational dynamics displayed by the diverse bCCP family members. As bCCP is an enzyme that is on the front-line of the native defenses of NIH Select List pathogens incuding Pseudomonas aeruginosa, Burkholderia complex speices, Vibrio cholarae, Campylobacter jejuni, Escherichia coli, Citrobacter, and Yersinia pestis, these studies will provide fundamental insight into the long-term development of new antimicrobial compounds that will target the novel features of bCCP structure.